Human immunodeficiency virus type 1 (HIV-1) is engaged in an ongoing evolutionary conflict with APOBEC3 (A3) proteins. Retrovirus-restricting A3?s (A3D, A3F, A3G, and A3H) gain access to progeny virions produced in infected cells primarily by binding RNAs to be packaged into virions. This positions the A3 enzymes for restricting virus replication in target cells by DNA deamination-dependent and -independent mechanisms. However, lentiviruses like HIV-1 encode an A3-counteraction protein called Vif (virion infectivity factor) responsible for degradation of these A3 enzymes. There are currently no antiretroviral therapies designed to promote A3 packaging or disrupt Vif-dependent A3 degradation. Our lab recently solved the crystal structure of human A3H and these high-resolution images revealed a novel and unanticipated RNA binding mechanism in which two A3H monomers become dimeric by complexing with duplex RNA. Moreover, the active site required for cytosine deamination occurs adjacent to the RNA-binding surface raising the possibility of RNA cytosine deamination (i.e. RNA editing). I will test the hypothesis that A3H binds to specific RNA structures in cellular and viral RNAs to restrict RNA-based viruses through deamination-dependent (RNA editing) and deamination- independent mechanisms through two specific aims. In Aim 1, I will identify the HIV-1 and cellular duplex RNA species bound by endogenous A3H through a series of A3H-RNA immunoprecipitation and deep sequencing experiments (RIP-seq). A3H-RNA interactions will be further interrogated by advanced imaging techniques including an in cellulo interaction protocol and single molecule fluorescence in situ hybridization. In Aim 2, I propose RIP-seq of A3H in infected with RNA-only viruses, including influenza A virus and poliovirus, to identify viral RNA structures bound and sites of RNA editing. I will determine if A3H changes the infectious titer of these viruses in single and multiple round infections. Finally, I will use fluorescent microscopy and RNA imaging to validate these A3H-RNA interactions. In summary, these proposed experiments use innovative technologies to improve our understanding of A3H-mediated restriction of HIV-1 as well as have the potential to open an entirely new research area of APOBEC-mediated riboviral restriction.